PROJECT SUMMARY While it is well established that estradiol (E2) -the main premenopausal ovarian hormone-, plays mitogenic roles in estrogen-receptor positive (ER+) breast cancer, emerging evidence shows that E2 promotes tumorigenesis and metastases of ER negative (ER-) tumors through effects on the microenvironment. E2 plays well known neurotrophic and neuroprotective functions in the adult brain by regulating the expression of neurotrophins, growth factors and chemokines, however, how E2 promotes brain metastatic progression remains ill-defined. The scientific premise of this proposal is that E2 promotes brain metastasis by modulating ER+ cells in the brain microenvironment. Our long-term goal is to define the mechanisms underlying the pro-metastatic effects of E2 in the brain to identify novel strategies to prevent or treat brain metastases. Our studies have shown that E2 promotes experimental brain metastases by modulating ER+ reactive astrocytes, which are induced early during brain metastatic colonization and are essential for brain metastatic progression. Reactive astrocytes surrounding brain metastases express both ER genes (ER? and ER?), and in response to E2, upregulate neurotrophins (BDNF) and growth factors (EGF) that activate pro- metastatic pathways (TRKB, EGFR, HER2) in cancer cells. E2 also represses chemokines (Ccl2, Ccl3, Ccl4, Ccl5), which promote recruitment and polarization of microglia into an M1 tumor-suppressive phenotype. Our central hypothesis is that E2 promotes brain metastases by at least two independent mechanisms: 1) upregulation of BDNF leading to activation of TRKB/EGFR signaling in metastatic cancer cells, and 2) repression of tumor-suppressive M1 microglia in the early brain metastatic niche. Therefore, targeting aromatase, E2/ER signaling or downstream BDNF/TRKB/EGFR activation can be used to prevent or treat brain metastases. In Aim 1 we will determine how E2 upregulates BDNF in the metastatic niche, and whether BDNF activation of TRKB/EGFR/HER2 is a mechanism of increased brain metastases in response to E2. In Aim 2 we will determine how E2 regulates microglia activation and polarization at early stages of brain metastases, and whether this is a mechanism of increased brain metastases in response to E2. In Aim 3 we will determine whether targeting E2/ER signaling or downstream BDNF/TRKB/EGFR activation reduces brain metastatic colonization and progression in vivo. This contribution will be significant because defining the mechanisms by which E2 modulates the brain niche could provide alternatives for prevention and treatment of brain metastases in women at high risk. This proposal is innovative because it represents a new paradigm in thinking about E2 and the brain niche in cancer; it will fill a critical gap by defining how E2 modulates microglia during brain metastasis and, it will define if aromatase inhibitors and/or ANA-12 have preclinical efficacy in prevention and treatment of TrkB+ brain metastases.